This invention relates to a method and apparatus to temporally regulate analysis of a nucleic acid.
For nucleic acid analysis of biological samples, the collected samples are routinely stored in various containers. After collection of the specimen, such as blood or hair, the container is usually sealed to isolate the specimen from the environment until analysis is performed. If analysis is postponed or if off-site analysis is to be performed, some samples may be maintained in a controlled environment in order to sustain specimen integrity. Further, the container must be rugged enough to withstand transit and storage without compromising the specimen. For example, samples collected at crime scenes must be accurately identified and described, and transported to a lab for subsequent analysis.
The potential for error in maintaining, labeling, handling, etc. a sample increases with the number of manipulative steps involved. Sample contamination is a concern since, as the specimen is processed, the sample is manipulated with the risk of identification error and/or sample contamination from exogenous nucleic acid, as nucleic acids are ubiquitous and contamination may occur from innocuous sources such as dust. Likewise, environmental contamination may be a concern, since a blood sample could contaminate the surrounding environment. Each time that the specimen is transferred, manipulated, etc., there is an opportunity to misidentify the specimen, contaminate the specimen or contaminate the environment. In addition, the container or specimen could be potentially misidentified or misplaced with each handling step, resulting in incorrect or incomplete sample identification.
Biological samples often are subjected to nucleic acid analysis to identify the source of the sample, or to identify one or more features of a sample. A standard analytical tool for nucleic acid analysis of specimens is the polymerase chain reaction (PCR). The basis of PCR is that it exponentially multiplies the quantity of nucleic acid extracted from a specimen to generate a quantity sufficient for analysis. PCR requires multiple reagents appropriate for amplification, such as a buffer, all four deoxyribonucleoside triphosphates (dNTP) (i.e., adenine triphosphate or dATP, thymine triphosphate or dTTP, cytosine triphosphate or dCTP, and guanine triphosphate or dGTP), magnesium, a polynucleotide polymerase enzyme, and at least one oligonucleotide primer.
Initially, nucleic acid is extracted from a specimen and added to the above reagents. The nucleic acid is denatured at a temperature between 90xc2x0 C. and 100xc2x0 C. to create two single-stranded polynucleotides that serve as templates. Primers then anneal to each nucleic strand at a temperature between 40xc2x0 C. and 75xc2x0 C. to demarcate a target sequence for the polymerase-catalyzed attachment of the appropriate dNTP. Repeated cycling of these processes (thermal cycling) results in quantities of nucleic acid sufficient for analysis.
Since PCR exponentially multiplies any nucleic acid present, whether endogenous or exogenous to the sample, samples to undergo PCR must be handled with the utmost care. Any exogenous nucleic acid present not only contaminates the specimen, but will be amplified during PCR, yielding an incorrect analysis. Since any contaminant nucleic acid is amplified, it is easy to appreciate why even trace contamination of the sample is fatal to obtaining an accurate result.
A unitary container is important for increasing the specificity of the PCR amplification and for increasing the shelf life of any pre-mixed PCR reagents in the container. U.S. Pat. No. 5,411,876 discloses a technique preparatory to PCR that interposes a barrier into the PCR reaction vessel. The barrier in the ""876 patent is composed of a hydrophobic substance, such as a grease or wax. One purpose of the barrier is to preserve reagent concentration during heating by attempting to prevent solvent evaporating from the PCR reaction tube. The melting point of the wax is chosen so that the less-dense molten wax floats on the surface of the solvent during PCR thermal cycling. After amplification, the solidified wax seals the reagents from the environment. If the PCR reaction tube is opened after amplification, the solidified wax barrier prevents both endogenous nucleic acid from escaping and endogenous nucleic acid from comingling with the endogenous nucleic acid. A disadvantage of this method, however, is that the addition of the hydrophobic material adds a handling step that poses a risk of contamination.
A second purpose of the technique disclosed in the ""876 patent is to pre-fill a PCR reaction vessel with reagents necessary for DNA amplification in a batch process. Batch processing removes a handling step by eliminating the necessity of adding the hydrophobic material during PCR thermal cycling, enables more accurate control over the addition of reagents, and creates a stockpile of PCR reaction tubes for future use. A wax barrier segregates one or more necessary reagents from the remaining reagents. The melting point of the wax is chosen such that the wax is solid at storage temperatures of about 0xc2x0 C. to 5xc2x0 C. Heating the PCR reaction tube above about 40xc2x0 C. causes the wax to melt, releasing the sequestered reagent and initiating the PCR process. Segregation of selected PCR reagents inhibits primer oligomerization and extends the stability of the reagent mixture from only a few days to a week or more. The segregation also minimizes annealing of primers to non-target sequences, which reduces the yield of amplified target sequences. If reagents are combined prior to the start of thermal cycling, mispriming occurs in an admixture of the nucleic acid to be amplified.
A limitation of the invention disclosed in the ""876 patent is that the disclosed vessel is not intended for prolonged storage of the contained reagents near room temperature. In addition, the container and method cannot be used with the intact biological specimen to be analyzed, but is limited to use with the nucleic acids that have already been extracted from the biological specimen, requiring at least one manipulation step, during which contamination or error can occur. Still another limitation is that the PCR reaction tube must be sized to fit the PCR instrument(s).
It is known to pre-fill sample containers with a reagent to conveniently process a specimen added to the container. To control exposure of the specimen added to the container, the reagent can be enclosed within a secondary frangible container contained within the primary sample container. The reagent is then released by rupture of the frangible container, and is subsequently mixed with the sample while confined within the primary sample container. One disadvantage of this method is that fragments from the frangible container can contact and hence contaminate the sample. Another disadvantage of this method is the addition of a costly manufacturing step to produce the intricate xe2x80x9ccontainer within a containerxe2x80x9d assembly. Although some pre-filled sample containers eliminate the frangible barrier, these are usually not amenable for use in an analytical instrument.
Thus, what is ideally desired is a simple, portable and inexpensive method and apparatus to allow the user to temporally regulate the analysis of a specimen containing a nucleic acid.
The inventive method and apparatus permit the collection, transport and identification of a specimen that contains nucleic acid, while also controlling exposure of the specimen to all reagents that will be needed for processing and analyses of the specimen using the polymerase chain reaction. By employing one or more breachable barriers to segregate one or more reagents from the specimen, the present invention permits temporal selectivity for processing and analyses. The present invention reduces fabrication costs by eliminating the need for a frangible barrier or a complicated container geometry. Many containers incorporating the present invention can be prepared by batch processing. This enables reproducible reagent compositions and reduces the cost and time involved to prepare individual containers.
Other advantages and features of the present invention will be appreciated with reference to the following drawings, detailed description, and examples.